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  • Ablation of vacuole protein sorting 18 (Vps18) gene leads to neurodegeneration and impaired neuronal migration by disrupting multiple vesicle transport pathways to lysoso ... 22854957

    Intracellular vesicle transport pathways are critical for neuronal survival and central nervous system development. The Vps-C complex regulates multiple vesicle transport pathways to the lysosome in lower organisms. However, little is known regarding its physiological function in mammals. We deleted Vps18, a central member of Vps-C core complex, in neural cells by generating Vps18(F/F); Nestin-Cre mice (Vps18 conditional knock-out mice). These mice displayed severe neurodegeneration and neuronal migration defects. Mechanistic studies revealed that Vps18 deficiency caused neurodegeneration by blocking multiple vesicle transport pathways to the lysosome, including autophagy, endocytosis, and biosynthetic pathways. Our study also showed that ablation of Vps18 resulted in up-regulation of β1 integrin in mouse brain probably due to lysosome dysfunction but had no effects on the reelin pathway, expression of N-cadherin, or activation of JNK, which are implicated in the regulation of neuronal migration. Finally, we demonstrated that knocking down β1 integrin partially rescued the migration defects, suggesting that Vps18 deficiency-mediated up-regulation of β1 integrin may contribute to the defect of neuronal migration in the Vps18-deficient brain. Our results demonstrate important roles of Vps18 in neuron survival and migration, which are disrupted in multiple neural disorders.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Interaction of AMSH with ESCRT-III and deubiquitination of endosomal cargo. 16760479

    The class E vacuolar protein sorting (VPS) pathway mediates sorting of ubiquitinated cargo into the forming vesicles of the multivesicular bodies (MVB), and it is essential for down-regulation of signaling by growth factors and budding of enveloped viruses such as Ebola and HIV-1. Work in yeast has identified DOA4 as a gene that is recruited by the class E machinery to remove ubiquitin from the endosomal cargo before it is incorporated into MVB vesicles, but the identity of the mammalian counterpart is unclear. Here we report the interaction of AMSH (associated molecule with the SH3 domain of STAM), an endosomal deubiquitinating enzyme, with the endodomal sorting complex required for transport (ESCRT-III) subunits CHMP1A, CHMP1B, CHMP2A, and CHMP3. We also show that a catalytically inactive AMSH inhibits retroviral budding in a dominant-negative manner and induces the accumulation of ubiquitinated forms of an endosomal cargo, namely murine leukemia virus Gag. Finally, VPS4 and AMSH compete for binding to the C-terminal regions of CHMP1A and CHMP1B, revealing a coordinated interaction with ESCRT-III. Taken together, these results are consistent with a role of AMSH in the deubiquitination of the endosomal cargo preceding lysosomal degradation.
    Document Type:
    Reference
    Product Catalog Number:
    AP106P
    Product Catalog Name:
    Rabbit Anti-Goat IgG Antibody, HRP conjugate

  • GGA proteins mediate the recycling pathway of memapsin 2 (BACE). 15615712

    Memapsin 2 (BACE, beta-secretase) is a membrane-associated aspartic protease that initiates the hydrolysis of beta-amyloid precursor protein (APP) leading to the production of amyloid-beta (A beta) and the progression of Alzheimer disease. Both memapsin 2 and APP are transported from the cell surface to endosomes where APP is cleaved by memapsin 2. We described previously that the cytosolic domain of memapsin 2 contains an acid cluster-dileucine motif (ACDL) that binds the VHS (Vps-27, Hrs, and STAM) domain of Golgi-localized gamma-ear-containing ARF-binding (GGA) proteins (He, X., Zhu, G., Koelsch, G., Rodgers, K. K., Zhang, X. C., and Tang, J. (2003) Biochemistry 42, 12174-12180). Here we report that GGA proteins colocalize in the trans-Golgi network and endosomes with memapsin 2 and a memapsin 2 chimera containing a cytosolic domain of a mannose-6-phosphate receptor. Depleting cellular GGA proteins with RNA interference or mutation of serine 498 to stop the phosphorylation of ACDL resulted in the accumulation of memapsin 2 in early endosomes. A similar change of memapsin 2 localization also was observed when a retromer subunit, VPS26, was depleted. These observations suggest that GGA proteins function with the phosphorylated ACDL in the memapsin 2-recycling pathway from endosomes to trans-Golgi on the way back to the cell surface.
    Document Type:
    Reference
    Product Catalog Number:
    AB5352
    Product Catalog Name:
    Anti-Amyloid Precursor Protein Antibody, CT

  • Alterations in the levels of vesicular trafficking proteins involved in HIV replication in the brains and CSF of patients with HIV-associated neurocognitive disorders. 24292993

    Human immunodeficiency virus (HIV) associated neurocognitive disorders (HAND) remain prevalent despite improved antiretroviral therapies. A HAND-specific biomarker indicative of neuropsychological impairment (NPI) would give insight into disease progression and aid clinicians in designing therapy. Endosomal sorting complex required for transport (ESCRT) proteins such as tumor susceptibility gene (TSG)-101, vacuolar protein sorting (VPS)-4 and LIP-5 are important for HIV replication and recently antiviral interferon stimulated gene (ISG)-15 was proposed as a biomarker for CNS injury. Here, we analyzed a well-characterized cohort of HIV+ cerebral spinal fluid (CSF) and postmortem brain specimens for multiple vesicular trafficking proteins and a related innate immune protein, ISG-15, TSG-101, VPS-4 and LIP-5. All protein levels trended higher with increased NPI and neuropathology. ISG-15 CSF levels were increased in HIV encephalitis (HIVE) compared to normal cases, and three quarters of HIVE samples had above average CSF ISG-15 levels. VPS-4 CSF levels were increased in NPI/NPI-O compared to normal patients. VPS-4 CSF levels in HIV-associated dementia were equivalent to that of normal patients. LIP-5 CSF levels positively correlate with ISG-15 levels, and higher than average ISG-15 levels indicate elevated viral load. Immunoblot and immunohistochemical analyses show increased expression of ISG-15, VPS-4 and LIP-5 in neuronal cell bodies and astroglial cells. ESCRT protein CSF levels analyzed in conjunction with viral load may be indicative of NPI stage, and may aid in the diagnosis and design of therapies for HIV patients. Further studies on the ESCRT protein expression during HIV infection may lead to a promising biomarker for predicting progression of NPI.
    Document Type:
    Reference
    Product Catalog Number:
    MAB1501
    Product Catalog Name:
    Anti-Actin Antibody, clone C4

  • Parvovirus B19 VP2-proteins produced in Saccharomyces cerevisiae: comparison with VP2-particles produced by baculovirus-derived vectors. 16316399

    The capsids of human parvovirus B19 consist of two structural proteins, the minor-capsid protein VP1 and the major-capsid protein VP2. The latter which constitutes for 95% of the capsid are able to form virus-like particles (VLPs) in yeast without the presence of VP1-proteins. VP2-proteins produced in Saccharomyces cerevisiae have the capacity to form VLPs in the absence of VP1-proteins. These yeast-derived VLPs resemble native virus or recombinant VP2-VLPs produced by baculovirus systems in respect of size, molecular weight and of antigenicity as shown by antigen-capture ELISA and T-cell proliferation tests. Regarding costs, yield and ease of handling particle production in yeast represents an alternative to the recombinant baculovirus expression system which is so far the source for VP2-VLPs of human parvovirus B19.
    Document Type:
    Reference
    Product Catalog Number:
    MAB8293
    Product Catalog Name:
    Anti-Parvovirus B19 Antibody, aa 328-344 of VP2 capsid protein, clone R92F6

  • Recombinant viral-like particles of parvovirus B19 as antigen carriers of anthrax protective antigen. 16724664

    Viral-like particles (VLPs) of parvovirus B19 were employed as an antigen carrier to present antigenic determinants of Bacillus anthracis. The small-loop peptide and the full-length domain 4 of protective antigen (PA) were chosen as immunogens for presentation on the VLP-capsid surface and subsequent immunization of BALB/c mice. The recombinant VLPs induced anti-PA IgG titers of up to 2.5 x 10(4). Neutralization assays showed that the recombinant VLPs elicited neutralizing anti-PA antibody titers of up to 1:400 and showed potential for the prevention of lethal toxin-induced mortality of mouse-macrophage cells (RAW264.7). In postimmune sera, no anti-PA titers were detected against synthetic small-loop peptide. Recombinant VLPs demonstrated the capacity to retain the immunogenicity of the displayed microbial PA-epitopes and elicited robust levels of anti-PA antibody titers. These findings suggest that the recombinant VLPs of parvovirus B19 have potential as an additional tool in the development of sub-unit vaccines.
    Document Type:
    Reference
    Product Catalog Number:
    MAB8293
    Product Catalog Name:
    Anti-Parvovirus B19 Antibody, aa 328-344 of VP2 capsid protein, clone R92F6

  • Monitoring human parvovirus B19 virus-like particles and antibody complexes in solution by fluorescence correlation spectroscopy. 14977050

    Fluorescence correlation spectroscopy (FCS) was used in monitoring human parvovirus B19 virus-like particle (VLP) antibody complexes from acute phase and past-immunity serum samples. The Oregon Green 488-labeled VLPs gave an average diffusion coefficient of 1.7 x 10(-7) cm2 s(-1) with an apparent hydrodynamic radius of 14 nm. After incubation of the fluorescent VLPs with an acute phase serum sample, the mobility information obtained from the fluorescence intensity fluctuation by autocorrelation analysis showed an average diffusion coefficient of 1.5 x 10(-8) cm2 s(-1), corresponding to an average radius of 157 nm. In contrast, incubation of the fluorescent VLPs with a past-immunity serum sample gave an average diffusion coefficient of 3.5 x 10(-8) cm2 s(-1) and a radius of 69 nm. A control serum devoid of B19 antibodies caused a change in the diffusion coefficient from 1.7 x 10(-7) to 1.6 x 10(-7) cm2 s(-1), which is much smaller than that observed with acute phase or past-immunity sera. Thus, VLP-antibody complexes with different diffusion coefficients could be identified for the acute phase and past-immunity sera. FCS measurement of VLP-immune complexes could be useful in distinguishing between antibodies present in acute phase or past-immunity sera as well as in titration of the VLPs.
    Document Type:
    Reference
    Product Catalog Number:
    MAB8292
    Product Catalog Name:
    Anti-Parvovirus B19 Antibody, clone 521-5D

  • Virtual electrode theory explains pacing threshold increase caused by cardiac tissue damage. 14726298

    The virtual electrode polarization (VEP) effect is believed to play a key role in electrical stimulation of heart muscle. However, under certain conditions, including clinically, its existence and importance remain unknown. We investigated the influence of acute tissue damage produced by continuous pacing with strong current (40-mA, 4-ms biphasic pulses with 4-Hz frequency for 5 min) on stimulus-generated VEPs and pacing thresholds. A fluorescent optical mapping technique was used to obtain stimulus-induced transmembrane potential distribution around a pacing electrode applied to the ventricular surface of a Langendorff-perfused rabbit heart (n = 5). Maps and pacing thresholds were recorded before and after tissue damage. Spatial extents of electroporation and cell uncoupling were assessed by propidium iodide (n = 2) and connexin43 (n = 3) antibody staining, respectively. On the basis of these data, passive and active three-dimensional bidomain models were built to determine VEP patterns and thresholds for different-sized areas of the damaged region. Electrophysiological results showed that acute tissue damage led to disappearance of the VEP with an associated significant increase in pacing thresholds. Damage was expressed in electroporation and cell uncoupling within an approximately 1.0-mm-diameter area around the tip of the electrode. According to computer simulations, cell uncoupling, rather than electroporation, might be the direct cause of VEP elimination and threshold increase, which was nonlinearly dependent on the size of the damaged region. Fiber rotation with depth did not substantially affect the numerical results. The study explains failure to stimulate damaged tissue within the concepts of the VEP theory.
    Document Type:
    Reference
    Product Catalog Number:
    MAB3068

  • A novel system for efficient gene transfer into primary human hepatocytes via cell-permeable hepatitis B virus-like particle. 16317706

    Protein transduction domains (PTDs) have been used to deliver a variety of biologically active cargo across cellular membranes. However the potential of PTDs to mediate transport of nanoparticular structures into the cytoplasm bypassing the endosomal compartment remains unclear. Cell-permeable virus-like particles (VLPs) harboring a marker gene based on hepatitis B virus nucleocaspids were established. Cell permeability was achieved by fusion with translocation motif (TLM)-PTD. Electron and confocal microscopy revealed that these VLPs translocate as complete particles across the plasma membrane and transverse the cytoplasm toward the nucleus. Inhibition of endocytosis did not affect translocation of these VLPs into the cytoplasm. Based on these particles, a gene transfer system was developed. To this end the particles were loaded with DNA-encoding small hepatitis B virus surface antigen (SHBs) or green fluorescence protein (eGFP) that served as marker genes. Although the DNA-packaging efficiency was very low, applying the appropriate number of VLPs to primary human hepatocytes a gene transfer efficiency of approximately 95% was observed. In conclusion, the TLM-PTD has the potential to mediate efficient transfer of assembled particles and its cargo, nucleic acids, into primary human hepatocytes. This provides the basis for development of novel transducible therapeutic or diagnostic particles.
    Document Type:
    Reference
    Product Catalog Number:
    MAB16990
    Product Catalog Name:
    Anti-Hepatitis B Virus Antibody, core Antigen (ayw), clone 14E11, a.a. 134-140

  • Is there a trigger role of peroxynitrite in the anti-arrhythmic effect of ischaemic preconditioning and peroxynitrite infusion 21699894

    This study has examined whether peroxynitrite (PN), generated during the preconditioning (PC) procedure or administered by brief intracoronary infusions, plays a trigger role in the anti-arrhythmic effects of preconditioning and peroxynitrite in anaesthetized dogs. To achieve this we infused the peroxynitrite scavenger uric acid (UA; 0.2mg/kg/min, i.v.) over a 30min period, just prior to a 25min occlusion of the left anterior descending coronary artery, in preconditioned (UA+PC, n=8), peroxynitrite-treated (UA+PN, n=8) and in control (UAC; n=9) dogs. The effects were compared to those obtained from groups (PC, n=10; PN, n=10; C1, n=14) without uric acid administration. Severities of ischaemia (ST-segment elevation, inhomogeneity of electrical activation) and ventricular arrhythmias (VPBs, VT, VF), plasma nitrate/nitrite levels, as well as myocardial superoxide and nitrotyrosine productions were determined. Both preconditioning and the infusion of peroxynitrite increased nitrotyrosine formation which was abolished by the simultaneous administration of urate. Despite this, the protective effects of preconditioning (i.e. reductions in arrhythmias, superoxide and nitrotyrosine productions, as well as the increase in nitric oxide availability), occurring during the prolonged period of occlusion and reperfusion were still present. In contrast, urate completely abolished the protection resulted from peroxynitrite administration. This effect is most probably due to the fact that urate has already scavenged peroxynitrite during the infusion. Interestingly, urate itself, given prior to ischaemia and reperfusion, was also protective. We conclude that peroxynitrite in nanomolar concentrations can induce an anti-arrhythmic effect but peroxynitrite, generated during the preconditioning stimulus, is not necessary for the preconditioning-induced anti-arrhythmic protection.Copyright © 2011 Elsevier B.V. All rights reserved.
    Document Type:
    Reference
    Product Catalog Number:
    MAB5404
    Product Catalog Name:
    Anti-Nitrotyrosine Antibody, clone 2A8.2